The Role of Reactive Species in Epileptogenesis and Influence of Antiepileptic Drug Therapy on Oxidative Stress.

2012·0.39 MB·English

by  Martinc, Bostjan

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328 Current Neuropharmacology, 2012, 10, 328-343
The Role of Reactive Species in Epileptogenesis and Influence of
Antiepileptic Drug Therapy on Oxidative Stress
Boštjan Martinc, Iztok Grabnar and Tomaž Vovk*
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
Abstract: Epilepsy is considered one of the most common neurological disorders. The focus of this review is the acquired
form of epilepsy, with the development process consisting of three major phases, the acute injury phase, the latency
epileptogenesis phase, and the phase of spontaneous recurrent seizures. Nowadays, an increasing attention is paid to the
possible interrelationship between oxidative stress resulting in disturbance of physiological signalling roles of calcium and
free radicals in neuronal cells and mitochondrial dysfunction, cell damage, and epilepsy. The positive stimulation of
mitochondrial calcium signals by reactive oxygen species and increased reactive oxygen species generation resulting from
increased mitochondrial calcium can lead to a positive feedback loop. We propose that calcium can pose both,
physiological and pathological effects of mitochondrial function, which can lead in neuronal cell death and consequent
epileptic seizures. Various antiepileptic drugs may impair the endogenous antioxidative ability to prevent oxidative stress.
Therefore, some antiepileptic drugs, especially from the older generation, may trigger oxygen-dependent tissue injury.
The prooxidative effects of these antiepileptic drugs might lead to enhancement of seizure activity, resulting in loss of
their efficacy or apparent functional tolerance and undesired adverse effects. Additionally, various reactive metabolites of
antiepileptic drugs are capable of covalent binding to macromolecules which may lead to deterioration of the epileptic
seizures and systemic toxicity. Since neuronal loss seems to be one of the major neurobiological abnormalities in the
epileptic brain, the ability of antioxidants to attenuate seizure generation and the accompanying changes in oxidative
burden, further support an important role of antioxidants as having a putative antiepileptic potential.
Keywords: Antiepileptic drugs, antioxidants, calcium, epileptogenesis, mitochondria, oxidative stress, reactive species,
signalling.
INTRODUCTION they also serve as Ca2+ buffer. As a consequence of Ca2+
influenced ATP production in the mitochondria, reactive
Epilepsy is one of the most common and heterogeneous
oxygen species (ROS) are generated. It is supposed that Ca2+
neurological disorders, with an estimated prevalence of 40 to
ions, as well as ROS, under physiological conditions work as
50 million patients worldwide [1]. The incidence of epilepsy
positive effectors and cellular signalling molecules within
is particularly high in children under 5 years of age and in
mitochondrial signalling pathways, while their overload can
individuals older than 65 years [2, 3]. About one fourth of all
lead to mitochondrial dysfunction, oxidative stress and cell
cases develop before the age of five [4].
damage. Neuronal dysfunction or death could serve as the
Epilepsy or epileptic condition is defined by a state of propagating factor that may lead to the epileptic condition.
recurrent, spontaneous (unprovoked) seizures, which can be
This review is focused on acquired epilepsy and
convulsive or non-convulsive episodes. It is characterized by
comprehensively examines the evidence for the initial role of
synchronized abnormal electrical activity arising from a subtle disturbance in Ca2+ and ROS cellular balance in
group of cerebral neurons [3, 5, 6].
epileptogenesis. Furthermore, the influence of anticonvulsant
Nowadays, the increasing attention is paid to the possible therapy on endogenous ability to prevent oxidative stress
connection between oxidative stress that results in will be reviewed.
disturbance of physiological signalling roles of Ca2+ which is
ETIOLOGY OF EPILEPSY
associated with the increased production of free radicals,
mitochondrial dysfunction, cell damage and consequently Epilepsy can be either idiopathic or symptomatic. It is
epilepsy. estimated that up to 50% of epilepsy cases are symptomatic
or acquired, therefore associated with a previous
Mitochondria are well known as the main source of the
neurological insult [7]. Acquired form of epilepsy does not
cell's adenosine triphosphate (ATP). In numerous cell types
have a genetic link and becomes more common as people

age. It is more widespread than genetic or idiopathic form.
Idiopathic epilepsy is mainly due to genetic causes or

*Address correspondence to this author at the Chair of Biopharmacy and
developmental central nervous system (CNS) disorders and
Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Aškerčeva
malformations, that most often affect mitochondrial or ion
7, 1000 Ljubljana, Slovenia; Tel: [phone]; Fax: [phone];
E-mail: [email protected] channel function. There are many genetic factors that

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The Role of Reactive Species in Epileptogenesis and Influence Current Neuropharmacology, 2012, Vol. 10, No. 4 329
contribute to the increased neuronal excitability present in epileptogenesis is still controversial. Neuronal cell death can
most idiopathic epilepsies. Any mutation or defect in be presented either as a consequence of epileptic seizure
mitochondrial respiratory chain complexes, synapses, and only, or both, a cause and a consequence, as proposed by the
neurotransmitter receptors or in the voltage and ligand increasing evidence [3, 9].
channels may alter brain excitability and cause epileptic
Seizures can result in neuronal cell death through
seizures. Epilepsy is in most cases multifactorial. It often
dynamic processes that might include genetic factors,
arises in part from both, the genetic and the acquired factors.
excitotoxicity induced mitochondrial dysfunction, altered
Aetiology varies among various age groups, patient groups
cytokine levels, oxidative stress, and changes in plasticity or
and geographical locations. In general, congenital and
activation of some late cell death pathways [12-14]. It was
perinatal conditions are the most common causes of early
suggested that oxidative damage and consequently neuronal
childhood epilepsy onset; while in adults, epilepsy is more
cell death have common pathogenic processes that can
likely to be due to external non-genetic causes. However,
contribute to epileptogenesis and to initiation and
this distinction is by no means absolute.
propagation of spontaneous recurrent epileptic seizures [15].
The epilepsies are normally divided into ‘early’ (i.e.
Initial brain insult causes a cascade of structural,
seizures occurring within a week of the insult) and ‘late’ (i.e.
physiological, and biochemical changes, such as altered
chronic epilepsy developing later). Between the injury and
cerebral blood flow and vasoregulation, disruption of the
the onset of late epilepsy, there is normally a ‘latent period’.
blood brain barrier, increased intracranial pressure, focal or
During this period, most likely epileptogenic processes are
diffuse ischemic haemorrhage, inflammation, necrosis and
developing, which raises the possibility for neuroprotective
disruption of fiber tracts and blood vessels [16]. In
interventions to prevent later epilepsy.
posttraumatic epilepsy, for example, it was suggested that
THE DEVELOPMENT PHASES OF SYMPTOMATIC after intracranial haemorrhage, red blood cells break down
EPILEPSY and release haemoglobin (Hb) and iron ions. Consequently
ROS are generated by iron- or Hb-mediated reactions [16,
It is generally accepted that development processes of 17]. Increased production of ROS induces a cascade of
acquired epilepsy occur in three major phases: the acute cellular and molecular changes that might lead to
phase - injury, the latency phase - epileptogenesis, and hyperexcitability of neurons or to neuronal death [18]. The
finally the phase of spontaneous recurrent seizures - chronic
hypothesis of neuronal death caused or propagated epilepsy
epilepsy [8].
is further supported by the fact that surgical removal of a
The Acute Phase – Injury damaged hippocampus improves the condition of epilepsy
patients [19]. Therefore nowadays, accumulating evidence
Epileptogenesis in acquired epilepsy can be initiated by a suggests that neuronal cell death may be both a cause and a
number of types of brain lesions and these numerous consequence of epileptic seizures [3].
aetiologies may vary with age. Illnesses in the form of
tumours, infections, status epilepticus, childhood febrile The Phase of Spontaneous Recurrent Seizures - Chronic
seizures, stroke, hypoxia and neurodegenerative diseases, Epilepsy
increase the incidence of acquired epilepsy. Furthermore,
The major mystery that still has to be explained is how
status epilepticus, stroke, and traumatic brain injury are the initial injury can produce these long-term changes in
considered as the three major causes of brain injuries and neuronal excitability that lead to spontaneous recurrent
acquired epilepsy [7]. seizures of acquired epilepsy.
Sometimes CNS insults are associated with immediate
As discussed above, brain insult results in increased
seizures in the acute setting, but since epileptogenesis phase
oxidation of cellular macromolecules prior to the death of
is required, this does not necessarily mean that it will lead to
vulnerable neurons. At this stage endogenous antioxidant
acquired epilepsy [8]. defence system prevents seizure-induced neuronal death [20-
22]. Oxidative stress has been suggested to be a significant
The Latent Phase - Epileptogenesis
consequence of excitotoxicity that plays a critical role in
The process whereby an initial central nervous system epileptic brain damage. Excessive ROS production leads to
insult leads to the development of the epileptic condition is increased intracellular concentration of Ca2+ ions through
referred to as epileptogenesis. This transition processes may mechanisms discussed in the following section. Ca2+
take years or even decades after the initial insult. This might overload is involved in seizure-induced neuronal death, as
suggest that a progression of changes in response to the well as in necrosis and apoptosis [23].
initial insult is critical to the development of the epileptic
It is suggested, that during the injury phase of seizures,
condition [3, 9]. Furthermore, epilepsy is usually not a static
Ca2+ concentration may reach high levels. If concentrations
condition, but evolves over the lifespan [10].
are moderately elevated, neurons are subjected to long
Despite enormous progress in neuroscience in the last lasting neuroplasticity changes. However, in case of very
few decades, so far, the exact mechanism of epileptogenesis high Ca2+ concentrations, cell death is induced. Ca2+
is not known [11]. Among the events that occur in response concentration remains elevated during the latent phase.
to the initial insult, neuronal cell death has received Therefore, many second messenger effects, which can
significant attention as the propagating factor that may lead produce long-lasting plasticity changes in these neurons, are
to the epileptic condition. However, its actual role in initiated [7]. High Ca2+ concentration in the epileptic neurons
330 Current Neuropharmacology, 2012, Vol. 10, No. 4 Martinc et al.
remains elevated also during the chronic epilepsy phase, and are Ca2+ re-uptake pumps. Furthermore, there is also an
plays a role in maintenance of the spontaneous recurrent important plasma membrane Na+/Ca2+ exchanger [31]. Its
seizures. It can alter GABA receptor recycling which can function is also modulated by oxidation. Currently, its exact
A
serve as a possible mechanism for the effect of Ca2+ on role is still unknown, as ROS can both, stimulate and
altering neuronal excitability [24]. Furthermore, increased decrease its activity.
intracellular Ca2+ concentration can effect numerous cell
The stimulation of mitochondrial Ca2+ signals by ROS
physiological processes, including gene transcription, protein
and increased ROS generation resulting from increased
expression and turnover, neurogenesis, neuronal sprouting,
mitochondrial Ca2+ can lead to a positive feedback loop. So
and many others [25].
Ca2+ can pose both, a physiological and a pathological effect
SIGNALLING ROLES OF Ca2+ AND ROS of mitochondrial function.
ROS play an important role in cell signalling [26]. Oxidative Stress as a Disturbance of Physiological
Mitochondria are important players in maintaining cell Ca2+ Signalling Roles of Ca2+ and Free Radicals
homeostasis and serve as Ca2+ buffer. Physiological
increases in mitochondrial Ca2+ lead to the activation of Oxidative stress is defined as an imbalance between
the production of ROS on one side and endogenous
tricarboxylic acid (TCA) cycle enzymes including isocitrate
antioxidant and repair capacity on the other, in favour of the
dehydrogenase, α-ketoglutarate dehydrogenase, maleate
former [32].
dehydrogenase, as well as succinate dehydrogenase [27].
Their activation results in increased concentrations of As Ca2+ buffer, mitochondria can suffer Ca2+ overload,
reduced substrates for oxidative phosphorylation (NADH which may lead to free radical production. It is thought, that
and FADH2). Therefore, physiological increase in a delicate balance exists between moderate ROS production
mitochondrial Ca2+ results in enhanced respiratory chain to modulate physiological signalling, and overproduction of
activity, increased proton pumping and consequently ROS resulting from mitochondrial Ca2+ overload that
generation of ROS. The reciprocal interaction between Ca2+ ultimately leads to oxidative stress, cellular damage, and
modulated ROS production and ROS modulated Ca2+ eventually cell death. Ca2+overload triggers the opening of
signalling underlies the concept of ROS and Ca2+ crosstalk. the mitochondrial permeability transition (MPT) pores which
Ca2+ can alter ROS production by simply increasing is linked to apoptosis via the mitochondrial pathway or
metabolic rate via TCA cycle stimulation [28]. Moreover, necrosis due to mitochondrial damage [33]. This confirms
Ca2+ can activate nitric oxide synthase (NOS) and generate the complex interdependence between mitochondrial
nitric oxide. Nitric oxide has been shown to inhibit complex energy production, Ca2+ uptake, ROS generation, ROS
IV, which can in turn lead to ROS production at the Q0 site neutralisation, and redox signalling. While physiological
of complex III [29]. Nitric oxide has also been shown to increases in mitochondrial Ca2+ are beneficial for
elicit synaptic glutamate release and therefore contribute to metabolism, overload is detrimental to mitochondrial
further excitotoxicity in neighbouring cells [29]. Hence, function and can become pathological.
Ca2+ may stimulate oxidative phosphorylation electron flux
Possible Role of Free Radical Homeostasis Disturbance
on one side, and partial inhibition of the electron transport
in Epileptogenesis
chain on the other. Inhibition of electron transport chain
could lead to an increased possibility of electron slippage to The role of free radical homeostasis in neuronal disorders
oxygen. is of major interest, since cells in the CNS are particularly
Just as Ca2+ plays a role in the production of ROS, vulnerable to the harmful effects of ROS and reactive
cellular redox state can significantly modulate Ca2+ nitrogen species (RNS). In addition, antioxidant defence
mechanisms in CNS are particularly poor. This is especially
signalling. Moreover, protein redox state modulates many
important because the brain is rich in mitochondria and is
cellular processes. Changes in structure caused by oxidizing
characterised by high aerobic metabolic activity, high
amino acid residues can change the activity of enzymes and
ion transporters [30]. In the context of Ca2+ signalling, redox oxygen consumption, high ratio of membrane surface area to
state and ROS can stimulate, as well as inhibit Ca2+ cytoplasmic volume, high concentration of polyunsaturated
channels/pumps/exchangers, generally increasing Ca2+ fatty acids, and a neuronal network vulnerable to disruption
channel activity and inhibiting Ca2+ pumps [31]. Precisely, [34]. The brain is also rich in iron, and brain damage releases
iron ions capable of catalyzing free radical reactions [35].
ROS can directly, or indirectly through changes in cell redox
Furthermore, superoxide radicals can arise also from the
homeostasis, oxidize redox-sensing thiol groups on the
auto-oxidation of catecholamines and in the cytoplasm by
ryanodine receptors leading to channel stimulation.
enzymes, such as xanthine oxidase [36]. It is important to
Ryanodine receptors are the primary Ca2+ release channel
note that endogenous antioxidants and repair capacity
involved in the sarcoplasmic Ca2+ release. Furthermore, ROS
weaken with aging [32]. Among brain cells, neurons are
derived stimulation of inositol-1,4,5-triphosphate receptor
particularly vulnerable to oxidative insults due to low levels
channels and inhibition of sarco/endoplasmic reticulum
of antioxidant enzymes, especially catalase (CAT) and
ATPase and plasma membrane Ca2+ ATPase, results in
glutathione peroxidase (GPX), and of nonenzymatic
increased mitochondrial Ca2+ concentrations. Inositol-1,4,5-
antioxidants, namely vitamin E and glutathione (GSH) [37].
triphosphate receptor channels are the primary Ca2+
endoplasmic reticulum release channels, while sarco/ In the CNS there are two major contrary acting
endoplasmic reticulum and plasma membrane Ca2+ ATPase neurotransmitters: the excitatory acting glutamate and the
The Role of Reactive Species in Epileptogenesis and Influence Current Neuropharmacology, 2012, Vol. 10, No. 4 331
inhibitory acting γ-amino butyric acid (GABA). The as glutamate and aspartate, or to a decrease of inhibitory
excitatory glutamate, which could act toxically at higher neurotransmitters, such as GABA.
concentrations, is thought to be one of the major contributors
Glutamate is mainly present in the intracellular space.
in oxidative stress development [38].
Increased glutamate concentration in the extracellular
Oxidative stress is capable of damaging diverse cellular compartment can be toxic to neurons [47]. Increase in the
components or molecular targets, including nucleic acids, extracellular concentration of glutamate in the case of CNS
lipids, proteins, and carbohydrates [23]. Oxidation of injury or disease has been linked to a number of potential
monosaccharide sugars results in the formation of mechanisms including excessive release and impaired
oxaldehydes, which can contribute to protein aggregation cellular uptake. ROS generation can also support the
[39]. induction of seizure activity by direct inactivation of
glutamine synthetase (GS), thus permitting an excessive
Free radicals may trigger disruption of nucleinic acids.
increase in glutamate. Excessive glutamate receptor
They can break DNA strands or directly modify purine and
activation caused either by glutamate or glutamate receptor
pyridine bases, which leads to deletions and other mutations.
agonists can induce neurotoxicity, which is described by the
DNA damage activates the DNA repair enzyme poly-ADP-
term excitotoxicity. This is manifested in excessive
ribose polymerase-1 (PARP-1), which overactivation
stimulation of glutamate receptors, namely NMDA (N-
depletes its substrate, nicotinamide adenine dinucleotide
methyl-D-aspartate) and AMPA (α-amino-3-hydroxy-5-
(NADH), slowing the rate of glycolysis, electron transport,
methyl-4-isoxazolepropionic acid) receptors, and an
and ATP formation, eventually leading to functional
associated overwhelming increase in free cytosolic and
impairment or cell death [40]. There is also mtDNA, which mitochondrial Ca2+ concentration.
is even more vulnerable target for free radical damage
because of diminished repair mechanisms and lack of Sometimes Ca2+ can reach high levels in neuronal cells
histones and due to its vicinity to the site of ROS generation. already during the injury phase. It is anticipated that
mtDNA disruption may lead to mitochondrial dysfunction prolonged seizures, like status epilepticus, result in sufficient
resulting in disturbed cell function. Finally, RNA is the most production of ROS to overwhelm the endogenous
susceptible to oxidative damage, since it is single stranded, mitochondrial antioxidant defences [23].
not protected by hydrogen bonding, and less protected by More usually, during the injury phase, Ca2+ levels are
proteins. RNA damages may result in altered proteins or
insufficient to cause cell death. However, later during the
dysregulation of gene expression [41]. One of mitochondrial latent phase, Ca2+ remains elevated and initiates many effects
disorders is the myoclonus epilepsy with ragged red fibers
mediated by second messengers and consequently causes
(MERRF) syndrome that is linked to point mutations in the
long lasting changes in neurons, including their death (see
mitochondrial tRNALys gene [42, 43]. Partial seizures are Fig. 1). If neuronal cells survive latent phase, Ca2+ still
also frequently noticed in mitochondrial encephalopathy
remains elevated during chronic phase and therefore plays
with lactic acidosis and stroke-like episodes (MELAS)
unique role in maintaining spontaneous recurrent seizures
syndrome, which is associated with mutations in the
[7].
mitochondrial tRNALeu gene [44, 45].
Overall, all oxidative modifications may disturb the
Polyunsaturated fatty acids in lipoproteins and
function of enzymes, receptors, neurotransmitters, and
phospholipids of biological membranes are also highly structural proteins, and therefore could contribute to
susceptible to oxidative damage, which leads to lipid progressive cell decline, aberrant neuroplasticity changes,
peroxidation (LP). LP disrupts biological membranes and is and ultimately even cell death [23].
thereby highly deleterious to their structure and function
[46]. A large number of by-products are formed during this Mitochondrial Involvement
process, since unsaturated hydroperoxides generated by
The main mitochondrial function is the production of
peroxidation of polyunsaturated fatty acids can break down cellular energy in the form of ATP by mitochondrial
to form different reactive aldehydes; the most known is respiratory chain through oxidative phosphorylation by five
malondialdehyde (MDA). Reactive aldehydes can bind multienzyme complexes located in the mitochondrial inner
covalently to proteins, thereby altering their function and membrane [29]. Complexes I-IV are oxidoreductases which
inducing cellular damage [3]. participate in the transfer of electrons from reduced
substrates to oxygen and create an electrochemical proton
Of particular importance is also protein redox state, as
gradient across the mitochondrial inner membrane. Complex
already described above in the section describing signalling
roles of Ca2+ and ROS. Another possible consequence of V (ATP synthesis) couples proton reuptake with adenosine
increased ROS production is impairment of the Na+/K+- diphosphate (ADP) phosphorylation in the matrix to generate
ATP [48] (Fig. 2). In addition to the energy production,
ATPase activity, which normally maintains ionic gradients
mitochondria also play a crucial role in the maintenance
of neuronal membranes. These chemical and electrical
of intracellular Ca2+ homeostasis, neurotransmitter
gradients generate the background for electrical activity,
biosynthesis, generation of ROS and mechanisms of cell
which is essential for normal nervous system functions. A
decrease in Na+/K+-ATPase activity might decrease the death [48].
convulsive threshold and therefore additionally lead to an More recently, accumulating evidence suggests that
increase in the release of excitatory neurotransmitters, such mitochondria are also involved in acquired forms of
332 Current Neuropharmacology, 2012, Vol. 10, No. 4 Martinc et al.
↑ ATP
consumption
Mitochondrial
dysfunction Energy
depletion
Overstimulation of
Ca2+ signaling ↑Extracellular
pathways glutamate
↑ Cytos olic
Ca2+
Cell damage
Brain
insult

- status Free
epilepticus
radicals
- stroke
- traumatic Hyperexcitability Cell dea th by rapid
brain injury necrosisor delayed
apoptosis
- childhood
febrile
seizures
- hypoxia
Seizures

Fig. (1). A schematic presentation of proposed mechanism of cell damage and seizure generation in acquired epilepsy. Seizures or initial
brain insult lead to accumulation of free radicals. Seizures alone, or through cell damage, cause hyperexcitability and increased extracellular
glutamate concentration, which results through increased cytosolic Ca2+ concentration and consequently overstimulated Ca2+ signalling
pathways in mitochondrial dysfunction, increased ATP consumption and energy depletion. This is associated with cell damage and necrotic
or delayed apoptotic cell death. Cell death in itself is again considered as a cause of seizures. Beyond that, hyperexcitability may lead to
lower seizure threshold, which can result in propagation reaction.
epilepsy. Increase in mitochondrial oxidative stress and translocator (ANT) impairs the influx of adenosine
subsequent damage to cellular macromolecules have been diphosphate (ADP) into the matrix for ATP synthesis.
demonstrated following repeated or prolonged seizures like Oxidation of manganese superoxide dismutase (MnSOD)
status epilepticus and other injuries, such as hypoxic- can further compromise antioxidant capacity and lead to
ischemic insults, traumatic brain injury, or viral infection further oxidative stress [58]. Increased production of ROS
[23]. Impairment of mitochondrial function and increased directly down-regulates proteins of tight junctions and
ROS production has recently been demonstrated in human activates matrix metalloproteinases that contribute in the
[49-51] and in several animal models of epileptic seizures opening of the blood brain barrier [59]. This allows the entry
[23, 52-54]. of neurotoxins and inflammatory cells, which potentiates
already existing oxidative stress.
These changes strongly affect neuronal excitability and
synaptic transmission, which is proposed to be highly Oxidative stress emerges as a common underlying cause
relevant for seizure generation [3, 49]. Mitochondria could of blood brain barrier dysfunction [60]. If disrupted,
be involved in pathways leading to neuronal cell death, transport of molecules between blood and brain is altered in
characteristic for the areas of epileptogenesis. Status both directions. Increased oxidative mtDNA damage,
epilepticus induced in experimental models by kainic acid mitochondrial hydrogen peroxide production, and alterations
or pilocarpine, are known to activate programmed cell in the mitochondrial base excision repair pathway have been
death mechanisms [55]. Moreover, increased production of noted in the rat hippocampus after a period of three months
ROS is a feature of partially respiratory chain-inhibited following status epilepticus [15]. One of the consequences of
mitochondria [56]. oxidative damage is the release of mitochondrial cytochrome
Mitochondrial proteins can be modified by carbonylation, c into the cytosol, most probably by MPT pore. The MPT
nitration, S-glutathionylation, or S-nitrosylation. Function of pore is an assembly of pre-existing proteins of the inner and
many metabolic enzymes in the mitochondrial extracellular outer mitochondrial membrane formed as a high conductance
compartment, including ATPase, cytochrome c oxidase, channel. It allows Ca2+ to be released from the mitochondria
NADH dehydrogenase, and NADH oxidase, can be [61, 62]. MPT pore is formed by the apposition of the
consequently altered [57]. Oxidation of adenine nucleotide voltage-dependent anion channel (VDAC) in the outer
The Role of Reactive Species in Epileptogenesis and Influence Current Neuropharmacology, 2012, Vol. 10, No. 4 333

Fig. (2). Assumed role of mitochondria, Ca2+, and ROS in neuronal excitotoxicity and epileptogenesis. After increase of mitochondrial Ca2+
influx, mediated by various mechanisms, the generation of both ROS and RNS elicit downstream cell death signalling. The Ca2+ passage can
lead to propagation and amplification of mitochondrial Ca2+ overload. The mitochondrial respiratory chain is shown with complexes I, II, III,
334 Current Neuropharmacology, 2012, Vol. 10, No. 4 Martinc et al.
Fig. (2). contd….
IV, and V. ROS and RNS can react with thiol groups (-SH) and subsequently cause opening of the MTP pore and Bax pore. One of the
consequences is release of cyt-c across the outer membrane, which is the major initiation step in a cascade reaction of cell apoptosis. The cell
death pathway may switch between apoptosis and necrosis, in relation to the intracellular ATP availability.
Symbols legend:
Ac-CoA: Acetyl coenzyme A; ANT: Adenine nucleotide translocase; Cyt-c: Cytochrome c; FADH : Flavin adenine dinucleotide phosphate;
2
GSH: Reduced glutathione; I–V: complexes I–V; MnSOD: Manganese superoxide dismutase; MPT: mitochondrial permeability transition;
mtDNA: Mitochondrial deoxyribonucleinic acid; NAD: Nicotine adenine dinucleotide; NO: Nitric oxide; NOS: NO synthase; PARP: Poly-
ADP-ribose polymerase; PBR: Peripheral benzodiazepine receptor; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; VDAC:
Voltage-dependent anion channel.

membrane, the ANT in the inner membrane and the soluble [69]. In this way it could be capable of leading to chronic
matrix protein cyclophilin D [62]. The MPT pore is triggered acquired epilepsies.
by high Ca2+ concentration and other stimuli, including ROS,
Mitochondrial dysfunction has gained considerable
which may promote MPT pore by causing oxidation of thiol
interest as a potential source of ROS and consequently a
groups on the ANT. Likewise, ADP, reduced GSH, pyridine
cause of epileptic seizures, especially in therapy-resistant
nucleotide pool, and acidic pH are all inhibitors of MPT
forms of severe epilepsy.
opening [31].
Recent proteomic studies confirmed that certain
Overall, as all other processes, also MPT triggering by
mitochondrial components are altered following seizure
ROS is potentiated by increased Ca2+. Ca2+ is believed to be
activity and that brain injury may be explained by these
the coordinating signal to cytochrome c release. Bax, a
alterations [70]. Hence, mitochondria can be considered as a
proapoptotic member of the Bcl-2 family, interacts with
target for potential neuroprotective strategies in epilepsy.
MPT pore to induce permeability transition and cytochrome
c release which is a key event in apoptosis [63]. Besides ANTIEPILEPTIC DRUG THERAPY AND OXIDATIVE
cytochrome c, opening of the MPT also leads to STRESS
mitochondrial depolarization and loss of matrix solutes
Antiepileptic drugs (AEDs), at least in part, impair
including GSH, ADP/ATP, etc. Cytochrome c released into
antioxidant systems. The ability of antioxidants to attenuate
the cytoplasm, further triggers the complex intrinsic
seizure generation and the accompanying changes in
mitochondrial pathway of apoptosis as shown in Fig. (2).
oxidative burden, further support an important role of
The cell death pathway may switch between apoptosis and
antioxidants as having putative antiepileptic potential.
necrosis, depending on the availability of intracellular ATP.
Epilepsy is usually well controlled by presently available
Whereas apoptosis is the principal cell death pathway in the
drugs. However, in 20 to 30% of patients seizures are not
presence of sufficient ATP, overwhelming depletion of ATP
results in necrotic cell death. In general, mitochondrial Ca2+ controlled with available medication. The majority of these
patients suffer from focal form of epilepsy. The areas of
overload and consequent mitochondrial dysfunction appear
epileptogenesis in these cases are usually characterised by
to be combining factors in excitotoxicity. They are playing
cell loss. One of the most frequent and devastating forms of
key roles in cell death [64].
epilepsy involves the development of an epileptic focus in
Epileptic seizures can occur as a presenting sign of temporal lobe structures. While the granular cell layer is
mitochondrial dysfunction in the central nervous system. relatively preserved, the progressive loss of pyramidal cells
This is seen in the occurrence of epileptic seizures in of the CA1, CA3 and CA4 layers are the neuropathological
mitochondrial diseases arising from mutations in mtDNA or hallmarks. Progressive cell loss is suggested to be a major
nuclear DNA [49]. In addition, systemic administration of reason why seizures of temporal lobe origin become
mitochondrial toxins, such as 3-nitropropionic acid and particularly resistant to antiepileptic drug therapy at later
cyanide, inhibits the functions of the mitochondrial stages of the disease [71].
respiratory chain that can compromise cellular energy
metabolism and induce seizures in animal models [65, 66]. Since antiepileptic treatment, to a various extent impairs
These accumulating evidences implicate that both, mtDNA the intrinsic ability to counteract oxidative stress by reducing
mutations and exogenous mitochondrial toxins cause endogenous antioxidants or increasing free radical
mitochondrial respiratory chain dysfunction which is production, progression of epilepsy was investigated in
associated with at least some of the mechanisms of various clinical studies.
epileptogenesis. Recently, evidence for a more general
Influence of AEDs on Antioxidants and Markers of
involvement of mitochondria, also in sporadic forms of
Oxidative Stress in Patients with Epilepsy
epilepsy has been provided [67, 68]. Collectively,
experimental and human epilepsy studies prove that The majority of oxidative stress measurements in patients
mitochondria are intimately involved in pathways leading to with epilepsy have been performed on peripheral tissues,
neuronal cell death [49]. Therefore, increased mitochondrial such as plasma, serum or red blood cells. However, oxidative
oxidative stress and mitochondrial dysfunction may be the stress may originate from various sources in the body and
final common pathway that underlines all these neuro- peripheral measurements might not necessarily accurately
pathological conditions and contributes to epileptogenesis reflect the oxidative stress in the CNS. Nevertheless, studies
The Role of Reactive Species in Epileptogenesis and Influence Current Neuropharmacology, 2012, Vol. 10, No. 4 335
on rat models of FeCl /kainate brain injection induced with phenytoin was also associated with lower TAC and
3
seizures indicate that lipid peroxidation products were GSH. The role of carbamazepine (CBZ) in generating free
increased not only in the CNS, but also in blood [72, 73]. It radicals and consequently causing neuronal damage is not so
should be mentioned that also the reverse situation is evident. CBZ enhanced plasma LP in adolescent and adult
possible. In a study by Aytan et al., it has been established patients [86] and caused marked increase in serum total
that peripheral oxidative stress can change oxidative status of peroxide levels in children [80]. Lower TAC [86] and GSH
CNS. In a rabbit model, feeding with high cholesterol diet [98] in plasma, and GPX and CAT in erythrocytes were also
caused an increase in serum MDA, which clearly correlated observed [92]. In accordance with its pro-oxidative action,
with an increase in protein oxidation parameters in the brain decreased SOD [92] and increased 8-OHdG [96] and
[74]. nitrite/nitrate in erythrocytes were also observed. However,
there are also studies which do not confirm CBZ pro-
Increased activities of SOD [75, 76] and CAT [75, 77],
oxidative action [83, 86, 91, 95] and even studies with anti-
and decreased activities of GPX [75, 78] and glutathione
oxidative action [88, 93, 99].
reductase (GR) [79] was observed in numerous studies in
drug-naive patients with epilepsy. In a recent study, LP and Long-term use of certain AEDs has been proposed to
percentage haemolysis in patients with epilepsy was higher increase free radical formation and cause oxidative damage
compared to controls [79]. Furthermore, erythrocyte GR and in neurons [90, 99, 110]. As demonstrated in Table 1, this is
plasma ascorbate and vitamin A concentrations were lower particularly true for older AED generation, namely VPA,
[79]. In the majority of these studies also increased markers phenytoin and CBZ. Exacerbation of oxidative stress during
of lipid peroxidation were noted [75, 77-82]. treatment with AEDs could also be one of the reasons for
pharmacotherapy resistant epilepsy. These pro-oxidative
On the other hand, there are also studies, with very weak
effects might lead to enhancement of seizure activity through
(unchanged SOD, CAT, GPX and GR activities) [78, 79, 82-
increased hyperexcitability and/or the induction of neuronal
84] or even opposite (decreased lipid peroxidation markers)
damage, which can result in loss of AEDs efficacy or
[85] association of oxidative stress with epilepsy.
apparent functional tolerance and undesired side effects.
AEDs have diverse effects on the antioxidative system Such functional tolerance may lead to complete loss of AED
[86-88]. Some of them, especially from the older generation activity and cross-tolerance to other AEDs Tolerance
of AEDs (such as CBZ and VPA) may trigger oxygen- develops to some AED effects much more rapidly than to
dependent tissue injury by several mechanisms [89]. others. It may lead to attenuation of side effects and also to
Oxidative injury may play a role in the initiation and loss of efficacy and is reversible after discontinuation of
progression of epilepsy, therefore therapies aimed at drug treatment [111]. Experimental evidence indicates that
reducing oxidative stress may ameliorate tissue damage and almost all first-, second-, and third-generation AEDs lose
favourably alter the clinical course. Peroxidation of their antiepileptic activity during prolonged treatment,
membrane lipids caused by an increase in generation of free although to a different extent [111].
radicals or decrease in the activities of antioxidant defence
Many conventional anticonvulsants, namely
systems has been suggested to be critically involved in the
phenobarbital, CBZ, and VPA are metabolized to generate
seizure control.
reactive metabolites with capability of covalent binding to
Oxidative stress in patients with epilepsy treated with macromolecules [86, 112]. Therefore, the AEDs may not
AEDs was assessed by measuring serum, plasma, erythrocyte, only suppress the epileptic seizures but also elicit systemic
leukocyte or urine markers of macromolecular damage (MDA, toxicity. There are at least two potential mechanisms by
RCD, P-SH, 8-OHdG, 15F-2t-isoP) and antioxidative which the free radicals produced by oxidation of phenolic
enzyme activity (SOD, GPX, CAT, GR) (Table 1). metabolites could lead to toxicity, by generating ROS and/or
by covalent binding of their metabolic intermediates to
The role of valproic acid (VPA) in exacerbation of proteins or other vital macromolecules [89, 112, 113].
oxidative stress is supported by reduced total antioxidant
capacity (TAC) [86, 90] and enhanced total oxidative status Unfortunately, till now little is known about pro-
(TOS) [90], reported for patients treated with VPA. oxidative or neuroprotective effects of newer second and
Erythrocyte GPX [91, 92], GR [93] and serum Se [94], uric third generation AEDs. Oxcarbazepine (OXC) and
acid and albumin [80], which are considered endogenous levetiracetam (LEV) are classified as newer, second
enzymatic and non-enzymatic antioxidant molecules, were generation AEDs [106]. As seen in the Table 1, OXC
found to be reduced in children and adults treated with VPA. antiepileptic actions could be due to its neuroprotective
Furthermore, in studies in patients with epilepsy, treated with effects. Arhan et al. have shown that OXC decreases serum
VPA, increased levels of oxidative macromolecular damage lipid peroxidation and nitrite/nitrate concentrations [101]. On
markers, such as MDA [76, 86, 90, 91] and 15F-2t-isoP [95], the other hand, Ozden et al. found LEV to enhance lipid
which are markers of enhanced lipid peroxidation and peroxidation [108]. To evaluate possible neuroprotective
increased levels of 8-OHdG [90, 96], which is a marker of effects of newer AEDs more investigations need to be done
nucleic acid damage, were observed. Similarly, enhanced in clinical practice on patients or on animal models.
lipid peroxidation [80] and decreased SOD and GPX [92]
Effects of AEDs on Oxidative Stress Markers in Different
was seen in studies with phenobarbital.
Animal Models of Seizures and Epilepsy
Elevated levels of lipid hydroperoxide were observed in
The generation of seizures is associated with changes in
phenytoin treated patients with epilepsy [88, 97]. Treatment the intracellular levels of antioxidants and oxidants. Various
336 Current Neuropharmacology, 2012, Vol. 10, No. 4 Martinc et al.
Table 1. Effects of Antiepileptic Drugs on Antioxidants and Oxidative Stress Markers in Blood of Patients with Epilepsy
AED Findings Materials Subject References
Valproic acid LP ↑ Erc Childhood [76, 99]
↑ Plasma Adolescent and adult [86]
↑/-/- Serum Childhood [90, 91, 100]*/[101]
(15F-2t-isoP) ↑ Urine Childhood [95]
8-OHdG ↑ Serum Childhood [90]
↑ Leukocytes Adult [96]
TAC ↓ Serum Childhood [90]
↓ Plasma Adolescent and adult [86]
TOS/XO ↑/- Serum Childhood [90]/[101]
SOD ↓ Erc Adult [92]/[98]
↑/- Erc Childhood [89, 91]/[100]*
- Plasma Childhood [102]
GPX -/↓ Erc Adult [92]
↓/↑/↓a Erc Childhood [91, 99]/[93]*/[100]
↑/- Plasma/Serum Childhood [102]
↑ Plasma Adolescent and adult [86]
GR ↑a /↓ Erc Childhood [100]/[93]
Se -/↓a Plasma/Serum Childhood [99]/[94]
-/↑ Serum Adolescent and adult [86, 92]
Nitrite/nitrate ↑ Serum Childhood [103]
NO - Serum Childhood [86]
GSH ↓ Erc Childhood [100]*
- Plasma Adult [98]*
Phenytoin LP ↑/↑ Serum Adult/ Uncertain [88] / [97]
TAC ↓ Serum Uncertain [97]
SOD ↑ Serum Adult [88]
GSH ↓ Serum/Plasma Adult [88]/[103]*
Carbamazepine LP ↑ Plasma Adolescent and adult [86]
(15F-2t-isoP) - Urine Childhood [95]
(MDA) ↓ Erc Childhood [93]
- Serum Childhood [104, 105]
Total peroxide ↑ Plasma Childhood [80]
8-OHdG ↑ Leukocytes Adult [96]
TAC ↓ Plasma Adolescent and adult [86]
SOD -/↓ Erc Adult [92]
↑/- Erc Childhood [89]/[88, 103, 106]
The Role of Reactive Species in Epileptogenesis and Influence Current Neuropharmacology, 2012, Vol. 10, No. 4 337
Table 1. contd….
AED Findings Materials Subject References
Carbamazepine ↑ Serum Adult [88]
GPX -/↓ Erc Adult [92]
↑/- Erc Childhood [93, 106]*
- Plasma Adolescent and adult [86]
CAT ↓ Erc Adult [92]
Se - Erc/Plasma Adolescent and adult [86, 92]
Nitrite/nitrate ↑ Serum Children [103]
GSH ↓ Erc Childhood [100]*
↓ Plasma Adult [98]*
Phenobarbital Lipid ↑ Plasma Childhood [80]
hydroperoxide
SOD/GPX ↓ Erc Adult [92]
GPX/GR -/↑ Plasma Childhood/Adult [79, 107]/[35]
Levetiracetam 8-OHdG - Leukocytes Adult [96]
LP (15F-2t-isoP) ↑ Urine Adult [108]
Oxcarbazepine LP / Nitrite/nitrate ↓/↓ Serum Childhood [86]
GPX/SOD -/- Erc Adults [109]
a Only in patients with a severe adverse effect related to valproic acid therapy
* Healthy control; ↓ decreased; ↑ increased; - no significant changes observed; CAT: Catalase; Erc: Erythrocytes; GPX: Glutathione peroxidase; GR: Glutathione reductase; GSH:
Glutathione; LP – Lipid peroxidation; MDA: Malondialdehyde; NO: Nitric oxide; SOD: Superoxide dismutase; TAC: Total antioxidant capacity; TOS: total oxidative status; 8-
OHdG: 8-hydroxydeoxyguanosine; 15F-2t-isoP: 15-F(2t)-isoprostane.

experimental animal seizures models have been designed and both, to a model of acute induced seizures culminating with
investigated specifically for the evaluation of the role of a prolonged episode of status epilepticus, as well as to a
various AEDs on modulating oxidative stress markers. The model of chronic spontaneous seizures [126]. During status
sites of seizure generation are not uniform in various models. epilepticus induced by pilocarpine in combination with
lithium, hyper metabolism occurs with increased glucose
In numerous studies generation of free radicals was
consumption, which results in abnormal respiratory chain
increased following pentylenetetrazol (PTZ) kindling, due to
and neuronal damage [127]. As reported by Bellissimo et al.,
increased cytosolic Ca2+ concentrations. PTZ is best known
in pilocarpine model of epilepsy, in rats presenting status
for its use in screening antiepileptic drugs [104]. It is a
epilepticus or spontaneous seizures, decreased activity of
tetrazole derivate with convulsant actions in mice, rats, cats,
SOD and increased levels of hydroperoxides in the
and primates, presumably by impairing GABA-mediated
hippocampus were observed [128]. To follow the evolution
inhibition by action at GABA receptor. PTZ is considered a
of neuronal damage caused by oxidative stress, cellular
GABA antagonist.
activation (investigated by fos protein expression and
Blood and brain toxicity after PTZ induced seizures and glucose utilization) and stress response (investigated by heat-
kindling were investigated in a rat model [125]. LP levels of shock protein (HSP72), immunoreactivity, and acid fuchsine
plasma, erythrocyte, and brain cortex and brain microsomal staining) in lithium-pilocarpine seizure adult rat models of
fraction were increased by PTZ administration. Additionally, various duration, were analysed [129]. These investigations
increased serum NO levels and accumulation of hydroxyl proved that regions with the largest metabolic activation
radicals were reported, while plasma and brain enzymatic coincided with the highest stress response and were
and nonenzymatic antioxidants (GPX, vitamin E) were consequently most heavily damaged [129].
decreased.
In animal models of seizures and epilepsy, various
Another often used model is pilocarpin induced model of AEDs were investigated. For instance, topiramate (TPM),
seizures and epilepsy. Pilocarpine is a cholinergic agonist is a voltage-gated Ca2+ channel inhibitor. The effects of
commonly used to induce seizures and an epilepsy-like TPM administration on pentylenetetrazol-induced rat model
status in rodents. The pilocarpin model of epilepsy relates of seizures showed that lipid peroxidation levels in serum